Biosensor

ABSTRACT

A biosensor that can perform analysis based on a sample noninvasively collected from a human body is provided. The biosensor comprises an identification substance (38) that binds to a substance to be detected (40), and an electrode (16) charged with a charge of the identification substance (38), comprises an inhibitor (39) that inhibits a substance not to be detected (42) from attaching to at least one of the identification substance (38) and the electrode (16), and detects a change in a charge density of the electrode (16) caused by binding of the substance to be detected (40) to the identification substance (38).

RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 national phase application of PCTApplication PCT/JP2014/070823 filed Aug. 7, 2014 which claims priorityto Japanese Application No. 2013-165086 filed Aug. 8, 2013. The entirecontent of each is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to a biosensor.

BACKGROUND ART

In recent years, as biosensors, techniques in which living cells can benoninvasively utilized for analysis have been disclosed (for example,Patent Literature 1). Patent Literature 1 discloses a biosensor having astructure in which a detection surface that detects a change in thephysical properties of a negative charge is coated with phenylboronicacid groups that bind to a sialic acid sample (a cell itself or a sugarchain derived from a cell).

CITATION LIST Patent Literature

Patent Literature 1: Japanese Patent Laid-Open No. 2010-107496

SUMMARY OF INVENTION Technical Problem

However, in the biosensor described in the above Patent Literature 1,cells and the like are not invaded, but it cannot be said that a humanbody is not invaded when cells are collected. In other words, abiosensor that can further reduce the burden on a human body, forexample, a biosensor that can detect a substance to be detected, basedon tears, sweat, saliva, or the like, is desired. In this connection, inaddition to glucose as a substance to be detected, proteins such asalbumin are contained in tears and the like, and there is a fear thatthe proteins as noise decrease measurement sensitivity.

Accordingly, it is an object of the present invention to provide abiosensor that can perform analysis based on a sample noninvasivelycollected from a human body.

Solution to Problem

A biosensor according to the present invention is a biosensor comprisingan identification substance that binds to a substance to be detected,and an electrode charged with a charge of the identification substance,in which the biosensor comprises an inhibitor that inhibits a substancenot to be detected from attaching to at least one of the identificationsubstance and the electrode; the identification substance is in contactwith the electrode; the inhibitor is formed of a polymer compound havinga longer molecular chain than the identification substance; aself-assembled monolayer is formed on a surface of the electrode fromthe identification substance and the inhibitor; and the biosensordetects a change in a charge density of the electrode caused by bindingof the substance to be detected to the identification substance.

In addition, a biosensor according to the present invention is abiosensor comprising an identification substance that binds to asubstance to be detected, and an electrode charged with a charge of theidentification substance, in which the biosensor comprises an inhibitorthat inhibits a substance not to be detected from attaching to at leastone of the identification substance and the electrode; the biosensorcomprises a thin film provided on the electrode and formed of theidentification substance, and one or two or more inhibitor layers formedon the thin film and comprising the inhibitor; and the biosensor detectsa change in a charge density of the electrode caused by binding of thesubstance to be detected to the identification substance.

Further, a biosensor according to the present invention is a biosensorcomprising an identification substance that binds to a substance to bedetected, and an electrode charged with a charge of the identificationsubstance, in which the biosensor comprises an inhibitor that inhibits asubstance not to be detected from attaching to at least one of theidentification substance and the electrode; the identification substanceis bound to the inhibitor; and the biosensor detects a change in acharge density of the electrode caused by binding of the substance to bedetected to the identification substance.

Advantageous Effect of Invention

According to the present invention, the substance not to be detected canbe inhibited from binding to the identification substance or attachingto the surface of the electrode by the inhibitor, and therefore themeasurement sensitivity can be further improved. Therefore, thebiosensor can more reliably measure glucose concentrations based on asample noninvasively collected from a human body.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic view showing the entire configuration of abiosensor according to a first embodiment.

FIG. 2 is a schematic view showing the configuration of anidentification portion in the biosensor according to the firstembodiment.

FIG. 3 is a graph showing the relationship between glucose concentrationand gate voltage change in the biosensor according to the firstembodiment.

FIG. 4 is a schematic view showing the configuration of anidentification portion in a biosensor according to a second embodiment.

FIG. 5 is a schematic view showing the configuration of anidentification portion in a biosensor according to a modification of thesecond embodiment.

FIG. 6 is a schematic view used for the explanation of theidentification portion in the biosensor according to the secondembodiment.

FIG. 7 is a graph showing the relationship (1) between glucoseconcentration and gate voltage change in a biosensor according to thesecond embodiment.

FIG. 8 is a graph showing the relationship (2) between glucoseconcentration and gate voltage change in a biosensor according to thesecond embodiment.

FIG. 9 is a graph showing the relationship (3) between glucoseconcentration and gate voltage change in a biosensor according to thesecond embodiment.

FIG. 10 is a graph showing the relationship (4) between glucoseconcentration and gate voltage change in a biosensor according to thesecond embodiment.

FIG. 11 is a schematic view showing the configuration of anidentification portion in a biosensor according to a modification of athird embodiment.

FIG. 12 is a graph showing the relationship between glucoseconcentration and gate voltage change in a biosensor according to thethird embodiment.

DESCRIPTION OF EMBODIMENTS

Embodiments of the present invention will be described in detail belowwith reference to the drawings.

1. First Embodiment

(1-1) Entire Configuration

A biosensor 10 shown in FIG. 1 comprises an identification portion 12Aand a field effect transistor (FET) 14 as a detection portion. Thebiosensor 10 identifies glucose, as a substance to be detected,contained in a sample in the identification portion 12A and converts theidentified information to an electrical signal in the FET 14 to detectglucose concentration in the sample. Here, examples of the sample caninclude noninvasively collected samples, that is, sweat, tears, andsaliva as biological fluids other than blood. In addition to glucose,proteins such as albumin as substances not to be detected are containedin these samples.

The identification portion 12A comprises an electrode 16 and a receptor20A provided on the electrode 16. In the case of this embodiment, in theidentification portion 12A, a container 18 is formed by providing acylindrical wall portion on one side surface of the electrode 16, and anidentification substance and an inhibitor are contained in the container18. The electrode 16 can be formed of Au but can also be formed of, forexample, Ag or Cu. The receptor 20A is formed of a Self-AssembledMonolayer (SAM) comprising an identification substance and an inhibitor.The SAM usually refers to an organic thin film in which at the interfacebetween a solid and a liquid or the interface between a solid and a gas,organic molecules gather together spontaneously and spontaneously form amonolayer.

The identification substance has the function of binding to glucosecontained in a sample. For the identification substance, phenylboronicacid can be used, and in addition, for example, derivatives thereof (forexample, phenylboronic acid having a vinyl group), and glucose-bindingproteins (GBPs) and derivatives thereof can be used. For example,phenylboronic acid produces a negative charge when binding to glucose.

The inhibitor inhibits a protein such as albumin, a substance not to bedetected, from binding to phenylboronic acid or attaching to the surfaceof the electrode 16. In the case of this embodiment, the inhibitor isformed of a polymer compound. For the polymer compound, oligoethyleneglycol having a longer molecular chain than the identification substancecan be used, and in addition, for example, polyethylene glycol can alsobe used.

As shown in FIG. 2, one end of each of an identification substance 38and an inhibitor 39 adsorbs on one side surface of the electrode 16 toform an SAM. In the identification substance 38 and the inhibitor 39, athiol group (—SH) or a disulfide group (—S—S—) is introduced to formderivatives of thiols or disulfides. Such derivatives of thiols ordisulfides can form a high density thin film on the surface of a metalsuch as Au, Ag, or Cu. For example, phenylboronic acid into which athiol group is introduced forms a strong bond such as Au—S. Theidentification substance 38 binds to glucose at the other end. Theinhibitor 39 specifically binds to a substance not to be detected at theother end.

The FET 14 comprises a source 24 and a drain 26 formed on a surface of asemiconductor substrate 22, and a gate insulating film 28 formed on thesemiconductor substrate 22, the source 24, and the drain 26 (FIG. 1).For the FET 14, both n-MOS and p-MOS can be used. A metal electrode 30is formed on the gate insulating film 28. The metal electrode 30 iselectrically connected to the electrode 16 via wiring 31. The metalelectrode 30 can be formed of Au, Ag, Cu, or the like.

The semiconductor substrate 22 may be formed of Si, Ga, As, ITO, IGZO,IZO, or the like, or an organic semiconductor, a carbon semiconductor(for example, carbon nanotubes, a graphene semiconductor, or a diamondsemiconductor), or the like may be used. The gate insulating film 28 canbe formed of an oxide or a nitride such as SiO₂, Si₃N₄(SiN_(x)), Ta₂O₅,or Al₂O₃.

A power supply 34 and an ammeter 36 are electrically connected to thesource 24 and the drain 26, and thus the drain current flowing from thesource 24 to the drain 26 can be measured. When the charge density onthe gate insulating film 28 changes, the magnitude of the drain currentchanges. In other words, in order to keep the drain current constant, itis necessary to change gate voltage with a change in charge density onthe gate insulating film 28. By measuring a change in the gate voltageof the FET 14, a change in charge density on the gate insulating film 28is electrically measured.

At this time, a reference electrode 32 may be used as shown in thisfigure. The reference electrode 32 is the electrode 16 that hasreference potential in the FET 14, and is electrically connected to theidentification substance 38 in the identification portion 12A.

(1-2) Manufacturing Method

The identification portion 12A shown in FIG. 2 can be manufactured bythe following procedure. First, Cr and Au are deposited in this order ona glass substrate using a sputtering apparatus to form the electrode 16.Then, a cylindrical wall portion formed of glass is fixed on theelectrode 16 with an epoxy resin followed by washing treatment using amixed solution of sulfuric acid and hydrogen peroxide and furtherwashing with pure water and ethanol in this order.

Then, a mixed liquid obtained by mixing an ethanol solvent comprising 1mM oligoethylene glycol (Hydroxy-EG₆-undecanethiol) and an ethanolsolvent comprising 1 mM 4-mercaptophenylboronic acid at a ratio of 9:1is put in the container 18. By holding this state for a predeterminedtime, the oligoethylene glycol and the phenylboronic acid chemisorb onthe surface of the electrode 16, and a self-assembled monolayer isformed. Finally, the mixed liquid is removed followed by washing withethanol and pure water in this order. The identification portion 12A canbe manufactured in this manner.

(1-3) Action and Effect

In the biosensor 10 formed as described above, first, a sample is addedto the identification portion 12A (FIG. 2). Glucose 40 contained in thesample reaches the lower part of the receptor 20A and binds to theidentification substance 38. Thus, the identification substance 38produces a negative charge. The surface of the electrode 16 is chargedwith the negative charge. On the other hand, a protein 42 such asalbumin contained in the sample binds to the inhibitor 39 and isinhibited from reaching the lower part of the receptor 20A, that is, theidentification substance 38, or the surface of the electrode 16.

The electrode 16 is electrically connected to the metal electrode 30 ofthe FET 14, and therefore when the surface of the electrode 16 ischarged with the negative charge, the charge density on the gateinsulating film 28 changes. A change in gate voltage accompanying thechange in charge density on the gate insulating film 28 of the FET 14 ismeasured. Thus, the biosensor 10 can detect the concentration of theglucose contained in the sample.

In this connection, the protein 42 has a negative charge and thereforeincreases the negative charge with which the surface of the electrode 16is charged by binding to the identification substance 38 or attaching tothe surface of the electrode 16. Thus, conventional biosensors have theproblem of a significant decrease in measurement sensitivity.

In the case of this embodiment, in the biosensor 10, the protein 42 isinhibited from reaching the identification substance 38 or the surfaceof the electrode 16 by the inhibitor 39 contained in the receptor 20A.Thus, in the biosensor 10, the protein 42 can be inhibited from bindingto the identification substance 38 or attaching to the surface of theelectrode 16, and therefore the electrode 16 can be inhibited from beingcharged with an unnecessary negative charge. Therefore, in the biosensor10, the measurement sensitivity can be further improved, and thereforethe glucose concentration can be more reliably measured based on asample noninvasively collected from a human body.

(1-4) Relationship Between Glucose Concentration and Gate Voltage Change

Next, a biosensor comprising the identification portion shown in FIG. 2was manufactured by the procedure shown in the above “(1-2)Manufacturing Method.” In the identification portion, phenylboronic acidwas used as an identification substance, and oligoethylene glycol wasused as an inhibitor. Then, a sample comprising albumin was placed inthe identification portion, and further a change in the gate voltage ofthe field effect transistor when the glucose concentration was graduallychanged was measured.

For the sample, Phosphate buffered saline (PBS) having a pH of 7.4 andcomprising 4 g/L albumin was provided, and glucose was added theretostepwise in the range of 100 μM to 10 mM to increase the glucoseconcentration stepwise. The results are shown in FIG. 3.

In FIG. 3, the vertical axis shows gate voltage change (mV), and thehorizontal axis shows the logarithm (log) of glucose concentration. Thecorrelation coefficient was 0.992, and the slope was 19.761, and it wasconfirmed that a linear relationship was seen between the logarithm ofglucose concentration and the gate voltage change. In other words, itcan be said that the biosensor is not influenced by the noise of theprotein, and therefore the amount of gate voltage change increasescorrespondingly to the glucose concentration. From the above results, itwas confirmed that by using a receptor formed of a monolayer comprisingan identification substance and an inhibitor, an increase in negativecharge due to a protein was inhibited.

2. Second Embodiment

An identification portion 12B according to a second embodiment will bedescribed with reference to FIG. 4 in which like numerals refer to partscorresponding to those in FIG. 2. The identification portion 12Baccording to this embodiment is different from the identificationportion according to the above first embodiment in that anidentification substance 38 is not fixed to one side surface of anelectrode 16.

(2-1) Configuration of Identification Portion

A receptor 20B contained in the identification portion 12B is formed ofa copolymer in which the identification substance 38 binds to aninhibitor 41. In the case of this embodiment, the receptor 20B furthercomprises a decomposition accelerator and a crosslinking agent.

The inhibitor 41 is formed of a hydrophilic polymer. The hydrophilicpolymer is a polymer having a hydrophilic functional group (a hydroxylgroup or a carboxyl group) and is a hydrogel, paper, a SuperabsorbentPolymer (SAP), or the like. In the case of this embodiment, a hydrogelis used for the inhibitor 41.

The hydrogel is a gel-like material in which hydrophilic polymer chainsare crosslinked to hold a large amount of water and which providesexcellent water absorbency. Examples of the hydrogel includepolyhydroxyethyl methacrylate (Poly-HEMA, also referred to aspoly2-hydroxyethyl methacrylate), polyvinylpyrrolidone (PVP), andpolyvinyl alcohol (PVA). The Poly-HEMA may be a homopolymer ofhydroxyethyl methacrylate (HEMA) or a copolymer with another monomer(for example, 2,3-dihydroxypropyl methacrylate or glycerol methacrylate(GMA)). The Poly-HEMA tends to have a higher water content when it is acopolymer. In addition, the PVP may be a homopolymer ofN-vinyl-2-pyrrolidone (NVP) or a copolymer obtained by using NVP as amain component and adding HEMA, methyl methacrylate (MMA), or the likeand a crosslinking agent for polymerization.

The paper is manufactured by gluing plant fibers or other fibers. Theplant fibers are composed of cellulose, hemicellulose, or lignin. Thecellulose has such a property that a large number of hydroxyl groups ofthe cellulose bind together by hydrogen bonds, and thus the plant fibersconstituting the paper stick together. In addition, examples of theother fibers include minerals, metals, and synthetic resins in the formof fibers. From the viewpoint of more firmly fixing the identificationsubstance 38, paper formed of plant fibers (cellulose) is preferred.

The SAP is a polymer that can absorb and hold water up to severalhundred times to several thousand times its own weight. As the SAP,polymers of acrylic acid can be used. The polymers of acrylic acid havea large number of carboxyl groups and therefore have highhydrophilicity, and form gels having high water absorbency when furthercrosslinked into fine structures and formed in the form of sodium salts.

Examples of other hydrophilic polymers can include cellulose derivativessuch as hydroxypropylmethylcellulose (HPMC), carboxymethylcellulosesodium (CMC-Na), and hydroxyethylcellulose (HEC); polysaccharides suchas alginic acid, hyaluronic acid, agarose, starch, dextran, and pullulanand derivatives thereof; homopolymers such as carboxyvinyl polymers,polyethylene oxide, poly(meth)acrylamide, and poly(meth)acrylic acid,copolymers of the homopolymers and polysaccharides or the like, andcopolymers of monomers constituting the homopolymers and other monomers;proteins such as collagen and gelatin and derivatives thereof; andpolysaccharides and mucopolysaccharides such as glycosaminoglycans suchas heparin, hyaluronic acid, chondroitin sulfate, dermatan sulfate,dextran sulfate, keratan sulfate, and heparan sulfate, chitin, andchitosan.

Further, hydrophilic polymers such as 1-vinyl-2-pyrrolidinone, 2-methylpropenoate ester, monomethacryloyloxyethyl phthalate, ammoniumsulfatoethyl methacrylate, N-vinylpyrrolidone, N,N-dimethylacrylamide,and 2-(methacryloyloxyethyl)-2-(trimethylammonioethyl) phosphate may beused.

The above illustrated hydrophilic polymers may be used singly, or two ormore types may be used in combination.

As the polymerization initiator, a known radical polymerizationaccelerator can be timely selected and used. Preferably, one havingwater solubility or water dispersibility and uniformly contained in theentire system is preferably used. Specifically, as the polymerizationinitiator, in addition to water-soluble peroxides, for example,potassium peroxodisulfate and ammonium peroxodisulfate, andwater-soluble azo compounds, for example, VA-044, V-50, and V-501 (allmanufactured by Wako Pure Chemical Industries, Ltd.), a mixture of Fe²⁺and hydrogen peroxide, and the like can be used.

As the crosslinking agent, N,N′-methylenebisacrylamide, ethylene glycoldimethacrylate, vinyl methacrylate, and the like can be used.

(2-2) Manufacturing Method

The identification portion 12B shown in FIG. 4 can be manufactured bythe following procedure. First, 0.15 g of 4-vinylphenylboronic acid, 1.0g of hydroxyethyl methacrylate, 0.5 g ofN-(3-dimethylaminopropyl)methacrylamide, and 0.05 g ofN,N′-methylenebisacrylamide as a crosslinking agent are provided, and6.0 g of a 6.7% by weight sodium acrylate aqueous solution (pH 7.3) isadjusted to a total amount of 10 g with ultrapure water. They are mixedfor dissolution in a container 18. Then, 25 μl oftetramethylethylenediamine and 7.5 mg of potassium peroxodisulfate aspolymerization initiators are added to initiate polymerization. Thisstate is held under a nitrogen atmosphere at room temperature for 2hours. After the completion of the polymerization reaction, the solutioncomprising the produced copolymer is immersed in ultrapure water toremove the unreacted components, and thus the receptor 20B in which theidentification substance 38 and the inhibitor 41 are copolymerized canbe obtained. The identification portion 12B can be manufactured in thismanner.

(2-3) Action and Effect

In the identification portion 12B formed as described above, thehydrophilic polymer, the inhibitor, has water molecules adsorbedtherearound and has high solvent affinity. Therefore, glucose comes intocontact with the hydrophilic polymer via the water molecules andtherefore dissolves in the solvent without being adsorbed. Thus, theglucose contained in the sample binds to the phenylboronic acid in thereceptor 20B, and thus a negative charge is produced, and the electrode16 is charged with the negative charge. Therefore, in the identificationportion 12B, an effect similar to that in the above first embodiment canbe obtained.

In addition, in the receptor 20B according to this embodiment, theidentification substance 38 binds to the inhibitor 41 formed of thehydrophilic polymer to form a copolymer. The hydrophilic polymer haswater molecules adsorbed therearound and has high solvent affinity.Therefore, a protein comes into contact with the hydrophilic polymer viathe water molecules and therefore dissolves in the solvent without beingadsorbed. Thus, in identification portion 12B, the protein contained inthe sample is prevented from binding to the identification substance 38or attaching to the surface of the electrode 16 by the inhibitor 41, andtherefore the measurement sensitivity can be further improved.Therefore, the biosensor can more reliably measure glucose concentrationbased on a sample noninvasively collected from a human body.

The inhibitor 41 may have a molecular template having the same structureas the molecular structure of glucose (not shown). The inhibitor 41having the molecular template can selectively take in the glucosecontained in the sample, and therefore the measurement sensitivity canbe further improved.

(2-4) Modification

An identification portion 12C according to a modification of the secondembodiment will be described with reference to FIG. 5 in which likenumerals refer to parts corresponding to those in FIG. 4. Theidentification portion 12C according to this embodiment is differentfrom the identification portion according to the above second embodimentin that an identification substance 38 is supported on a support 44.

A receptor 20C comprises the support 44, the identification substance 38supported on the support 44, and an inhibitor 41 and is formed of acopolymer in which the identification substance 38 binds to theinhibitor 41.

For the support 44, conductive particles and nonconductive particles canbe used. For the conductive particles, metal particles, for example,particles of Au, Pt, Ag, Cu, and the like, and nonmetal particles, forexample, particles of Indium Tin Oxide (ITO), conductive polymers, andthe like, can be used. In addition, for the nonconductive particles, forexample, particles of SiO₂ and the like can be used. For example, byintroducing a thiol group (—SH) or a disulfide group (—S—S—) intophenylboronic acid as an identification substance to form a derivativeof a thiol or a disulfide, the phenylboronic acid can be supported onthe surfaces of Au particles.

A procedure for manufacturing the receptor 20C will be described.Specifically, first, 9 ml of a gold nanocolloidal solution (5 nmdiameter,) and 1 ml of a 10 mM 4-mercaptophenylboronic acid(manufactured by Sigma-Aldrich Corporation)/ethanol solution are mixed,and allowed to stand at 25° C. for 24 hours to form a phenylboronicacid-gold nanoparticle solution. Next, 1.0 g of hydroxyethylmethacrylate (HEMA), 5 g of the above phenylboronic acid-goldnanoparticle solution, 0.5 g of N-3-(dimethylamino)propylmethacrylamide,3 g of a 6.7% by weight sodium acrylate aqueous solution (pH 7.3), and0.05 g of N,N′-methylenebisacrylamide are mixed, and adjusted withultrapure water so that the total amount is 10 g. Then, 5 mg ofpotassium peroxodisulfate and 5 μl of tetramethylenediamine aspolymerization initiators are added to initiate polymerization. Thisstate is held under a nitrogen atmosphere at room temperature for 2hours. After the completion of the polymerization reaction, the solutioncomprising the produced copolymer is immersed in ultrapure water toremove the unreacted components, and thus the receptor 20C in which theidentification substance 38 and the inhibitor 41 are copolymerized canbe obtained. The identification portion 12C can be manufactured in thismanner.

As shown in FIG. 6, some of the identification substance 38 supported onthe support 44 binds to the inhibitor 41 (numeral 45 in the figure) toform a copolymer. The remaining identification substance 38 supported onthe support 44 binds to glucose 40 contained in a sample. The glucose 40contained in the sample binds to the identification substance 38, andthus a negative charge is produced, and an electrode 16 is charged withthe negative charge. Therefore, an effect similar to that in the abovefirst embodiment can be obtained.

In addition, in the identification portion 12C according to thismodification, the identification substance 38 binds to the inhibitor 41formed of a hydrophilic polymer to form a copolymer, and therefore theprotein contained in the sample can be prevented from binding to theidentification substance 38 or attaching to the surface of the electrode16. Therefore, also in the identification portion 12C according to thismodification, an effect similar to that in the above second embodimentcan be obtained.

Further, in the identification portion 12C according to thismodification, the identification substance 38 is supported on thesupport 44, and therefore the identification substance 38 can also beeasily fixed particularly to paper.

(Relationship between Glucose Concentration and Gate Voltage Change)

Next, a biosensor comprising the identification portion shown in FIG. 4was manufactured by the procedure shown in the above “(2-2)Manufacturing Method.” For a sample, Phosphate buffered saline (PBS)having a pH of 7.4 and comprising 4 g/L albumin was provided, andglucose was added thereto stepwise in the range of 50 μM to 1.25 mM toincrease glucose concentration stepwise. The results are shown in FIG.7.

From FIG. 7, the correlation coefficient was 0.9959, and the slope was6.438, and it was confirmed that a linear relationship was seen betweenthe logarithm of glucose concentration and gate voltage change. From theabove results, it was confirmed that by using a copolymer in which anidentification substance bound to an inhibitor formed of a hydrophilicpolymer, an increase in negative charge due to a protein was inhibited.

Next, a biosensor comprising the identification portion shown in FIG. 5was manufactured by the procedure shown in the above “(2-4)Modification.” For the inhibitor in this case, hydroxyethyl methacrylatewas used. For a sample, Phosphate buffered saline (PBS) having a pH of7.4 and comprising 4 g/L albumin was provided, and glucose was addedthereto stepwise in the range of 50 μM to 1.25 mM to increase glucoseconcentration stepwise. The results are shown in FIG. 8.

From FIG. 8, the correlation coefficient was 0.9959, and the slope was6.438, and substantially the same results as FIG. 7 were obtained. Fromthe above results, it was confirmed that even if a copolymer in which anidentification substance supported on a support bound to an inhibitorformed of a hydrophilic polymer was used, an increase in negative chargedue to a protein was inhibited.

Further, a biosensor in which the inhibitor was changed to cellulose inthe identification portion shown in FIG. 5 was manufactured by theprocedure shown below. Specifically, first, 9 ml of a gold nanocolloidalsolution (5 nm diameter) and 1 ml of a 10 mM 4-mercaptophenylboronicacid (manufactured by Sigma-Aldrich Corporation)/ethanol solution weremixed, and allowed to stand at 25° C. for 24 hours to form aphenylboronic acid-gold nanoparticle solution. Next, 500 μl of the abovephenylboronic acid-gold nanoparticle solution was dropped on Kimwipes(registered trademark) cut to a length of 40 mm and a width of 10 mm,and dried at 60° C. The Kimwipes (registered trademark) after the dryingwas adhered to a gate electrode portion using a polydimethylsiloxanesolution (manufactured by Dow Corning Toray Co., Ltd.) to obtain amolecule identification member in which paper and phenylboronicacid-gold nanoparticles were mixed.

For a sample, Phosphate buffered saline (PBS) having a pH of 7.4 andcomprising 4 g/L albumin was provided, and glucose was added theretostepwise in the range of 10 μM to 2 mM to increase glucose concentrationstepwise. The results are shown in FIG. 9.

From FIG. 9, the correlation coefficient was 0.9846, and the slope was20.123, and it was confirmed that a linear relationship was seen betweenthe logarithm of glucose concentration and gate voltage change. From theabove results, it was confirmed that even when an identificationsubstance supported on a support was fixed to an inhibitor formed ofcellulose, an increase in negative charge due to a protein wasinhibited.

In addition, a biosensor in which a molecular template was formed in theinhibitor in the identification portion shown in FIG. 4 was manufacturedby the procedure shown below. First, 0.2 g of hydroxyethyl methacrylate(HEMA), 0.1 g of N-3-(dimethylamino)propylmethacrylamide, 0.01 g ofvinylphenylboronic acid, 0.02 g of N,N′-methylenebisacrylamide, 300 μlof 6.7% by weight sodium acrylate (pH 7.3), and 0.009 g of glucose wereadjusted to a total amount of 1 g with a 100 mM sodium phosphate buffer(pH 10.0) and dissolved, and then 10 μl of potassium peroxodisulfate (50mg/ml, manufactured by Wako Pure. Chemical Industries, Ltd.) and 2 μl oftetramethylenediamine (manufactured by TOKYO CHEMICAL INDUSTRY CO.,LTD.) as polymerization initiators were added to make a monomersolution.

Next, 15 μl of the monomer solution was dropped on an electrode 5 mmsquare formed of gold, covered with a PET (polyethyleneterephtalate)film, and subjected to a polymerization reaction under a nitrogenatmosphere at room temperature for 12 hours to make a hydrogel on theelectrode. After the completion of the polymerization reaction, the gateelectrode was immersed in a 0.1 M hydrochloric acid/methanol solutionovernight to remove the monomer components and glucose to form areceptor in which an identification substance and an inhibitor werecopolymerized. In this manner, a biosensor according to the embodimentin which the electrode surface was covered with a receptor was made.

As a comparison, the same receptor as the above was formed in an area 5mm square on an electrode 10 mm square formed of gold to make abiosensor in which part of the electrode was exposed.

1500 μl of a 100 mM sodium phosphate buffer (pH 9.0) was dropped on thereceptor of the biosensor made, and a gate voltage of 1 V and asource-drain current of 700 μA were made constant by a FET real timemeasurement apparatus. In this state, a change in gate electrode surfacepotential when 15 μl of 1 M glucose and 15 μl of a 100 mg/ml albuminsolution were added to the receptor was measured.

The results are shown in FIG. 10. In FIG. 10, the vertical axis showsgate voltage (V), and the horizontal axis shows time (seconds). In thisfigure, the solid line is the results of the biosensor according to theabove embodiment, and the broken line is the results of the biosensormade as a comparison.

From this figure, in the biosensor according to the embodiment, the gatesurface potential when 10 mM glucose is added changes in the negativedirection. From this, it was confirmed that in the biosensor accordingto the embodiment, a response to glucose was obtained. In addition, inthe biosensor according to the embodiment, the gate surface potentialdid not change even if albumin was added. From this, it was confirmedthat in the biosensor according to the embodiment, the inhibitorinhibited an increase in negative charge due to the protein. On theother hand, in the biosensor made as a comparison, the gate surfacepotential changed when albumin was added. This is considered to be dueto the fact that the albumin binds to the electrode.

3. Third Embodiment

An identification portion 12D according to a third embodiment will bedescribed with reference to FIG. 11 in which like numerals refer toparts corresponding to those in FIG. 2. The identification portion 12Daccording to this embodiment is different from the identificationportion according to the above first embodiment in that an inhibitor isformed on an identification substance 38 in the form of a layer.

A receptor 20D comprises a thin film 46 formed of the identificationsubstance 38, and an inhibitor layer 47 formed on the thin film 46 andformed of an inhibitor.

The thin film 46 is an SAM formed by the adsorption of one end of theidentification substance 38 on one side surface of an electrode 16. Theinhibitor layer 47 is formed of a hydrophilic polymer comprising ahydrogel, a SAP, or the like. In the case of this embodiment, theinhibitor layer 47 is formed of hydroxyethyl methacrylate.

The thin film 46 can be made by forming a self-assembled monolayer as inthe procedure shown in “(1-2) Manufacturing Method” in the above firstembodiment. Specifically, a gold substrate is immersed in a 1 mM4-mercaptophenylboronic acid (manufactured by Sigma-AldrichCorporation)/ethanol solution at 25° C. for 24 hours to make aself-assembled monolayer.

The inhibitor layer 47 is made by the following procedure. 1.0 g ofhydroxyethyl methacrylate (HEMA), 0.5 g ofN-3-(dimethylamino)propylmethacrylamide, 6 g of a 6.7%; by weight sodiumacrylate aqueous solution (pH 7.3), and 0.05 g ofN,N′-methylenebisacrylamide are mixed, and adjusted with ultrapure waterso that the total amount is 10 g. Then, 5 mg of potassiumperoxodisulfate and 5 μl of tetramethylenediamine as polymerizationinitiators are added to initiate polymerization. This state is heldunder a nitrogen atmosphere at room temperature for 2 hours. After thecompletion of the polymerization reaction, the solution comprising theproduced copolymer is immersed in ultrapure water to remove theunreacted components, and thus the inhibitor layer 47 can bemanufactured.

Finally, the inhibitor layer 47 formed of hydroxyethyl methacrylate islaid on the thin film 46 formed of the identification substance 38, andthus the identification portion 12D can be manufactured.

In the identification portion 12D formed as described above, thehydrophilic polymer, the inhibitor, has water molecules adsorbedtherearound and has high solvent affinity. Therefore, glucose 40 comesinto contact with the hydrophilic polymer via the water molecules andtherefore dissolves in the solvent without being adsorbed. Thus, theglucose 40 binds to the identification substance 38, and a negativecharge is produced, and the electrode 16 is charged with the negativecharge. Therefore, in the identification portion 12D, an effect similarto that in the above first embodiment can be obtained.

In addition, in the receptor 20D according to this embodiment, the thinfilm 46 of the identification substance 38 formed on the electrode 16 iscovered with the inhibitor layer 47. The hydrophilic polymer forming theinhibitor layer 47 has water molecules adsorbed therearound and has highsolvent affinity. Therefore, a protein comes into contact with thehydrophilic polymer via the water molecules and therefore dissolves inthe solvent without being adsorbed. Thus, in identification portion 12D,a protein 42 contained in a sample is prevented from binding to theidentification substance 38 or attaching to the surface of the electrode16 by the inhibitor layer 47, and therefore the measurement sensitivitycan be further improved. Therefore, the biosensor can more reliablymeasure glucose concentration based on a sample noninvasively collectedfrom a human body.

(Modification)

In the above third embodiment, a case where the number of inhibitorlayers is one has been described, but the present invention is notlimited to this, and two or more inhibitor layers formed of hydrophilicpolymers having different molecular weights may be formed.

(Relationship between Glucose Concentration and Gate Voltage Change)

Next, a biosensor comprising the identification portion shown in FIG. 11was manufactured by the above-described procedure. For a sample,Phosphate buffered saline (PBS) having a pH of 7.4 and comprising 4 g/Lalbumin was provided, and glucose was added thereto stepwise in therange of 10 μM to 2 mM to increase glucose concentration stepwise. Theresults are shown in FIG. 12.

From FIG. 12, the correlation coefficient was 0.9877, and the slope was25.94, and it was confirmed that a linear relationship was seen betweenthe logarithm of glucose concentration and gate voltage change. From theabove results, it was confirmed that by laying in the form of a layer aninhibitor formed of a hydrophilic polymer on a thin film formed of anidentification substance, an increase in negative charge due to aprotein was inhibited.

4. Modifications

The present invention is not limited to the above embodiments, andappropriate changes can be made within the spirit of the presentinvention. For example, in the case of the above embodiments, a casewhere the detection portion is a FET has been described, but the presentinvention is not limited to this, and light-receiving elements such asphotodiodes and photomultipliers, thermistors, Quartz CrystalMicrobalances (QCMs), elements utilizing surface plasmon resonance, andthe like can also be used.

In addition, a case where the identification portion and the detectionportion are electrically connected by wiring has been described, but thepresent invention is not limited to this, and the identification portionand the detection portion may be integrally formed. In other words, theelectrode may be directly formed on the gate insulating film of the FETas the detection portion.

REFERENCE SIGNS LIST

-   10 biosensor-   12A, 12B, 12C, 12D identification portion-   14 FET (detection portion)-   16 electrode-   28 gate insulating film-   30 metal electrode-   31 wiring-   38 identification substance-   39, 41 inhibitor-   40 glucose (substance to be detected)-   42 protein (substance not to be detected)-   44 support-   46 thin film-   47 inhibitor layer

The invention claimed is:
 1. A biosensor comprising an identificationsubstance that binds to a substance to be detected, and an electrode,wherein the biosensor comprises an inhibitor that inhibits a substancenot to be detected from attaching to at least one of the identificationsubstance and the electrode; the identification substance is in contactwith the electrode; the inhibitor is formed of a polymer compound havinga longer molecular chain than the identification substance; aself-assembled monolayer is formed on a surface of the electrode fromthe identification substance and the inhibitor; and the biosensordetects a change in a charge density of the electrode caused by bindingof the substance to be detected to the identification substance, whereinthe electrode is connected to a gate insulating film of a field effecttransistor, and wherein the electrode is disposed away from the fieldeffect transistor and electrically connected to a metal electrodeprovided on the gate insulating film via wiring.
 2. The biosensoraccording to claim 1, wherein the substance to be detected is glucose.3. The biosensor according to claim 1, wherein the identificationsubstance is phenylboronic acid.
 4. A biosensor comprising anidentification substance that binds to a substance to be detected, andan electrode, wherein the biosensor comprises an inhibitor that inhibitsa substance not to be detected from attaching to at least one of theidentification substance and the electrode; the biosensor comprises athin film provided on the electrode and formed of the identificationsubstance, and one or two or more inhibitor layers formed on the thinfilm and comprising the inhibitor; and the biosensor detects a change ina charge density of the electrode caused by binding of the substance tobe detected to the identification substance wherein at least one of theinhibitor layers is formed of homopolymers and/or copolymers ofpolyhydroxyethyl methacrylate (Poly-HEMA), polyvinylpyrrolidone (PVP),polyvinyl alcohol (PVA), 2,3-dihydroxypropyl methacrylate, glycerolmethacrylate (GMA), N-vinyl-2-pyrrolidone (NVP), methyl methacrylate(MMA), or any combination thereof.
 5. The biosensor according to claim4, wherein the electrode is connected to a gate insulating film of afield effect transistor.
 6. The biosensor according to claim 5, whereinthe electrode is disposed away from the field effect transistor andelectrically connected to a metal electrode provided on the gateinsulating film via wiring.
 7. The biosensor according to claim 4,wherein the identification substance is phenylboronic acid.
 8. Abiosensor comprising an identification substance that binds to asubstance to be detected, and an electrode, wherein the biosensorcomprises an inhibitor that inhibits a substance not to be detected fromattaching to at least one of the identification substance and theelectrode; the identification substance is supported on a conductive ornonconductive particles support and bound to the inhibitor, wherein theinhibitor comprises a hydrophilic polymer and molecular templates areformed in the inhibitor, wherein the molecular templates have astructure complementary to a molecular structure of the substance to bedetected, wherein the entire surface area of the electrode facing asample is covered with the inhibitor, wherein the hydrophilic polymer isa hydrogel, paper, or a Superabsorbent Polymer (SAP), wherein theconductive or nonconductive particles support comprises particles ofgold (Au), platinum (Pt), silver (Ag), indium tin oxide (ITO), SiO₂, orany combination thereof; and the biosensor detects a change in a chargedensity of the electrode caused by binding of the substance to bedetected to the identification substance.
 9. The biosensor according toclaim 8, wherein the electrode is connected to a gate insulating film ofa field effect transistor.
 10. The biosensor according to claim 9,wherein the electrode is disposed away from the field effect transistorand electrically connected to a metal electrode provided on the gateinsulating film via wiring.
 11. The biosensor according to claim 8,wherein the identification substance is phenylboronic acid.